Emily L. Lowe, Ph.D.
Microbiology, Immunology and
Molecular Genetics
UCLA

Models Systems to Study HIV:
In vitro and In vivo
• In vitro: Cell lines, primary cells
• In vivo: Humanized mice
• In vivo: Non-human Primates

Models Systems to Study HIV:
In vitro and In vivo
• Genetically modified viruses
– Modify the envelope to allow entry into more (or less) cell types
– Add a fluorescent protein to enable better visualization
• Green fluorescent protein (GFP), green
• mCherry, red
• SIV (Simian Immunodeficiency
Virus)
• SHIV: chimeras of HIV and SIV
Yu et al. HIV traffics through a specialized, surface-accessible intracellular compartment during trans-infection of T cells by mature
dendritic cells. PLoS Pathog. 4 (8). 2008. e1000134.

• In vitro: Taking place in a test tube, culture dish or elsewhere OUTSIDE a living organism
• Excellent tools for simple questions with limited variables
– Best for testing more focused approaches
• Cells are easily manipulated
• Cost efficient
• Scalable
• Excellent for initial drug screens

• Cancer cell lines are IMMORTALIZED
– Can be expanded in culture for long periods of time
– Are easily manipulated to express (or not) CD4, CCR5 and/or CXCR4 as well as host restriction factors
• “Primary cells” come from people or animal subjects
– Can purified to look at single or multiple populations
– Can only be maintained in culture for short periods
– Can be manipulated but not as easily as cancer cell lines

• To see HIV virions
• To see an HIV infected cell infect another cell
• To see where HIV goes in a cell
• To see what HIV interacts with in a cell
• Identify latency inducers

Cryofluorescence Light Microscope
170X 3500X 50,000X
June et al., Direct Visualization of HIV-with Correlative Live-Cell Microscopy and
Cry-Electron Tomography. Structure. 19 (11). 2011. 1573-1581.

June et al., Direct Visualization of HIV-with Correlative Live-Cell Microscopy and
Cry-Electron Tomography. Structure. 19 (11). 2011. 1573-1581.

Dr. Thomas Huser’s Lab http://cbst.ucdavis.edu/research/hiv-transmission-visualization

In vitro study to see where HIV goes in a cell it has infected
Campbell et al., Visualization of a proteasome-independent intermediate during
restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3). 2008. 549-561

In vitro study to see where HIV goes in a cell it has infected
Green = HIV
Red = “body”
Campbell et al., Visualization of a proteasome-independent intermediate during
restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3). 2008. 549-561

In vitro study to see what HIV interacts with inside a cell
Red = host restriction factor
Green = HIV
Orange = interaction!!!
Campbell et al., Visualization of a proteasome-independent intermediate during
restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3). 2008. 549-561

Kim et al., Recruitment of THIIH to the HIV LTR is a rate-limiting step in the
emergence of HIV from latency. EMBO J. 25. 2006. 3596-3604.

In vitro studies have given us a lot but…
• One major limitation to vaccine and therapeutic cure research has been the lack of an animal model that recapitulates all of the salient features of HIV-1 infection in humans.
• In vivo: Experimentation using a whole, living organism

• When considering species other than human as models for HIV-1 infection, the cellular proteins of the species must support viral replication.
• Humanized mice
• Non-human primates

In vivo Models to Study HIV:
Humanized Mice
Most importantly, these animals CAN be infected with HIV!
• Develop systemic viremia
• CD4 T cell loss

Akkina, R. New generation humanized mice for virus research: comparative aspects and future prospects. Virology.
435 (1). 2013. 14-28.

In vivo Models to Study HIV:
Humanized Mice
• Stem cells can be genetically manipulated (gene therapy)
• Not terribly cost effective
– Surgery requires technical skill
– Cannot be bred
– Special housing/handling due to immune compromised status
• Scalable allows for multiple “n”
– Can make up to 30 mice per tissue pair
– Excellent for initial drug-toxicity/efficacy screens
• IV and mucosal routes of infection possible
• Non-hematopoietic cells (lung, intestine mucosa) cannot be studied in organ systems

In vivo Models to Study HIV:
Non-human primates
• Many species of African monkeys and apes are natural hosts for SIV, but generally do not develop disease as a consequence of infection.

Natural Hosts (African)
African green monkey
SIVagm
©2011 by Cold Spring Harbor Laboratory Press
Sharp P M , and Hahn B H Cold Spring Harb Perspect Med
2011;1:a006841

In vivo Models to Study HIV:
Non-human primates
• By contrast, infection of Asian macaques, which are NOT natural hosts for primate lentiviruses, with certain strains of SIV results in high viral loads, progressive CD4+ T cell depletion and opportunistic infections.

Natural Hosts (African) Non-natural Hosts (Asian)
©2011 by Cold Spring Harbor Laboratory Press
Sharp P M , and Hahn B H Cold Spring Harb Perspect Med
2011;1:a006841

In vivo Models to Study HIV:
Non-human primates and SIV
HIV-1 group M, which is the most prevalent
HIV strain, jumped from chimpanzees into humans.
SIV cpz
HIV-1 http://www.awf.org/content/wildlife/detail/chimpanzee
HIV-2 originated in sooty mangabeys and is responsible for fewer infections than HIV-1
SIV smm
HIV-2 SIV mac http://pin.primate.wisc.edu/factsh eets/entry/sooty_mangabey

In vivo Models to Study HIV:
Non-human primates and SIV
• SIV: Simian Immunodeficieny Virus
– First isolated at the New England Primate Research
Center, Massachusetts, from rhesus macaques with a transmissible form of immunodeficiency characterized by opportunistic infections and tumours
– Later traced to an outbreak of lymphoma in the 1970s among macaques that were housed at the California
National Primate Research Center, California
– Monkeys might have received tissues from SIVinfected sooty mangabeys during experiments aiming to develop a non-human primate model for prion disease

In vivo Models to Study HIV:
Non-human primates and SHIVs
• SHIV
– Simian immunodeficieny virus containing HIV sequences/elements
– HIV-based vaccines cannot be tested with SIV
– SIV is not sensitive to many drugs that inhibit HIV-1
– SIV uses CD4 and CCR5 (like HIV-1) but my also use other co-receptors complicating testing of entry inhibitors
• Env-SHIV
• RT-SHIV
• stHIV-1

In vivo Models to Study HIV:
Non-human primates and SHIVs
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.

In vivo Models to Study HIV:
Non-human primates and SHIVs
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.

In vivo Models to Study HIV:
Non-human primates and SHIVs
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.
Pig tailed macaque

Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.

• Most commonly used
• Pathogenesis similar to
HIV-1
• High viral loads
• Progressive depletion of mucosal or peripheral
CD4+ T cells
• Destruction of lymph node architecture
• Progress AIDS faster (1-2 yrs)
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.

• Next most commonly used
• Lower viral loads and less
CD4+ T cell depletion
• Progress AIDS faster
(within 42 weeks)
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.

• Least used
• SIV strains are less pathogenic (probably because they are passaged in Rhesus macaques)
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio. 10. 2012. 852-867.

In vivo Models to Study HIV:
Non-human primates
• Stem cells can be genetically manipulated (gene therapy)
• Cost prohibitive
• Primate research contains ethical controvery
• Not scalable
• IV and mucosal routes of infection possible
• All tissues match (non-hematopoietic and immune) allowing for organ systems to be studied

Models to study HIV
Cats and Feline Immunodeficiency Virus
Wongsrikeao et al., Antiviral restriction factor transgenesis in the domestic cat.
Nature Methods. 8. 2011. 853-859.